Mitchell, N.J.; (2010) Application of chemically modified oligonucleotides in nanopore sensing and DNA nano – biotechnology. Doctoral thesis , UCL (University College London).

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Abstract

This thesis describes how targeted chemical modification can enhance the properties of nucleic acids for use in (i) nanopore analytics and (ii) nanobiotechnology. In nanopore analytics, individual molecules are detected as they pass a nanoscale pore to give rise to detectable blockades in ionic current. Despite progress in the sensing of a multitude of molecular species, the analytical resolution in the sensing of DNA is poor as individual bases in passing strands cannot be resolved due to the high speed of translocation. Here a new approach is presented which slows down single stranded DNA and enables the detection of multiple separate bases. Chemical tags are attached to bases, which cause a steric blockade each time a modified base passes a narrow pore. The resulting characteristic current signatures are specific for the chemical composition and the size of the tags. The unique electrical signatures can be exploited to encode sequence information as demonstrated for the discrimination between drug resistance-conferring point mutations. In addition, the generation of nucleotides with tailored properties may help develop a fast nanopore approach to size highly repetitive DNA sequences for forensic applications. In DNA nanobiotechnology, oligonucleotides are self-assembled via hybridization to generate higher-order structures of defined geometry. Here, the functional range of DNA nanostructures is expanded by chemically modifying the constituent nucleic acids. Firstly, tetrahedron-shaped nanostructures are demonstrated to act as a scaffold to assemble a multitude of different chemical groups at tunable stoichiometry and at geometrically defined sites. The new molecular entities exhibit functional properties beneficial in biosensing and diagnostics. In addition, an approach is presented to achieve self-assembly between DNA-strands via covalently attached tags that form reversible yet tight metal chelate complexes. This chemical strategy to form supramolecular structures can potentially be extended to protein or peptide networks of interest in basic science and technology.

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